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From Proteopedia

Revision as of 17:47, 21 April 2010

Overview

Xanthine oxidoreductase (XOR) is an extensively studied metalloflavoprotein that is found in a variety of different organisms, ranging from bacteria to eukaryotes. XORs are dimeric enzymes typically around 300 kDa in size with two interconvertible forms: xanthine dehydrogenase (XDH) [1.17.1.4] and xanthine oxidase (XO) [1.17.3.2]. Conversion between the two forms is mediated through the reversible oxidation of two cysteines residues or irreversible trypsin truncation. XOR is involved in purine catabolism, catalyzing the oxidation of hypoxanthine and xanthine to urate through the extraction of two electrons. The transport of these electrons is facilitated by the molybdenum of the molybdopterin co-factor, two iron sulfur centers, and a bound FAD coenzyme. In XDH the electrons are then passed preferentially from the reduced flavin to a final NAD+ acceptor, creating NADH. Apart from NADH, XDH may also use O2 as a final electron acceptor. In contrast, conversion to the XO form precludes NAD+ from binding, permitting only the use of O2. Consequently, the reduction of O2 produces substantial amounts of H2O2 and superoxide as byproducts. The production of these oxidative species has been implicated in the innate immune response ^[1] and cardiovascular disease, such as atherosclerosis ^[2], ischemia-reperfusion injury, and chronic heart failure ^{[3] [4]}.

Mechanism

Xanthine Oxidation

Several mechanisms have been suggested for the oxidation of xanthine to urate by xanthine oxidoreductase. However, a substantial amount of data appears to favor a mechanism in which a deprotonated molybdenum hydroxyl attacks the C8 atom of xanthine. This mechanism begins with the extraction of a proton from the hydroxyl of the molybdenum center by Glu1261 ^[5], an event computed to occur readily in the presence of the substrate ^[6]. The electrons from the deprotonated oxygen are then free to attack the electrophilic C8 atom of the bound xanthine. The formation of glutamic acid stabilizes this structure through hydrogen bond interactions with the N1 atom ^[7] as well as possible interactions between Arg-880 of the active site and enolate tautomerization at C6 ^[8]. Bond formation between the substrate and the molybdenum center orients a Mo = S moiety equatorially to the substrate, positioning it favorably for a concomitant hydride transfer from xanthine N7 ^[9]. Extraction of this hydride produces Mo-SH and reduces the Mo center from Mo VI to Mo IV. This intermediate breaks down through electron transfer from the molybdenum center through the iron-sulfur clusters, known as Fe-S I and Fe-S II to the bound FAD, forming FADH2. In this mechanism the Fe-S clusters function as electron sinks, maintaining an oxidized Mo-cofactor and a reduced FADH2. The Mo atom serves as a transducer between the two electrons passed from the substrate to the single electron of system of the Fe-S clusters. The transfer of electrons can be monitored through the formation of the paramagnetic transient Mo V ^[10]. Subsequent reduction of NAD+ to NADH in the case of xanthine dehydrogenases and O2 to H2O2 regenerates the oxidized FAD. Other mechanisms involving protonated molybdenum hydroxyls have been proposed with similar calculated activation energies (40 kcal/mol). However, the products in these cases have been computationally determined to be less stable that the reactant complex ^[11].